Bicarbonate binding activity of the CmpA protein of the cyanobacterium Synechococcus sp strain PCC 7942 involved in active transport of bicarbonate

The cmpABCD operon of the cyanobacterium Synechococcus sp, strain PCC 7942 encodes an ATP-binding cassette transporter involved in HCO3- uptake. The t hree genes, cmpBCD, encode membrane components of an ATP-binding cassette t ransporter, whereas cmpA encodes a 42-kDa cytoplasmic membrane protein, whi ch is 46.5% identical to the membrane-anchored substrate-binding protein of the nitrate/nitrite transporter. Equilibrium dialysis analysis using H14CO 3-, showed that a truncated CmpA protein lacking the N-terminal 31 amino ac ids, expressed in Escherichia coli cells as a histidine tagged soluble prot ein, specifically binds inorganic carbon (CO2 or HCO3-). The addition of th e recombinant CmpA protein to a buffer caused a decrease in the concentrati on of dissolved CO2 because of the binding of inorganic carbon to the prote in. The decrease in CO2 concentration was accelerated by the addition of ca rbonic anhydrase, indicating that HCO3-, but not CO2, binds to the protein. Mass spectrometric measurements of the amounts of unbound and bound HCO3- in CmpA solutions containing low concentrations of inorganic carbon reveale d that CmpA binds HCO3- with high affinity (K-d = 5 mu M). A similar dissoc iation constant was obtained by analysis of the competitive inhibition of t he CmpA protein on the carboxylation of phosphoenolpyruvate by phosphoenolp yruvate carboxylase at limiting concentrations of HCO3-. These findings sho wed that the cmpA gene encodes the substrate-binding protein of the HCO3- t ransporter.